Protein A/G Magnetic Beads for IP

Advantages

• Spinning free, IP (takes<30 minutes) with minimal sample loss.
• Background caused by non-specific binding is very low.
• Beads contain 9.3×1013 molecules/cm2 of protein A/G. Antibody binding capacity up to 0.6-0.8 mg/ml.
• Cheap to 73 USD/mL

Price Comparison

Properties

Reagent

Supplied in 25mM Tris-HCl (pH7.2), 50% StabiGuard, containing 0.01% (w/v) sodium azide.

Storage & stability

Protein A/G Magnteic Beads for IP should be stored in 50% glycerol at 2-8 ℃ for maximum stability. The unopened product is stable for one year when stored as indicated. After use, the resin should be cleaned and stored in 25mM Tris-HCl (pH 7.2), 50% StabiGuard, containing 0.01% (w/v) sodium azide. Do not freeze in the absence of glycerol.

Specificity

Protein A and Protein G have different binding selectivities depending on the origin of the IgG. Use Table 1 to select Protein A MagBeads or Protein G MagBeads according to the source and sub-type of the specific antibody.

Description

Protein A/G Magnteic Beads for IP use a biological nanosurface technology (S-TEC). Protein A/G is orientated as a coat on the surface of super paramagnetic microspheres with high coating density up to 9.3 × 1013 molecules/cm2. Compared to other similar immune magnetic beads, Biotool MagBeads™ Protein A/G display more antibody binding sites, therefore during IP, less magnetic beads are used. Non-specific binding is low, enabling Biotool MagBeads Protein A/G to be used in IP conveniently and efficiently. With a large, specific surface area, these beads can greatly shorten the equilibrium antibody and antigen adsorption time, enabling complete antibody antigen adsorption process within 10 minutes, and complete total purification and precipitation in just 30 minutes. This product can be used on a wide variety of samples, such as in cell lysates, supernatants collected from cell secretion, serum, ascites, and other immune antigens.

Procedure

This procedure (Figure 1) offers a general guideline for immunoprecipitation (IP). Optimization may be required for each antibody and target antigen. Protein A/G Magnteic Beads for IP are ideally suited for IP reactions.

1 Preparation of Protein A/G Magnteic Beads Matrix

1.1 Resuspend the Magnteic Beads in the vial (vortex >30 sec or tilt and rotate for 2 min).

1.2 Transfer 50 μl (the amount may be scaled up or down as required) Protein A/G Magnteic Beads to a 1.5 mL tube. Place the tube on the magnet to separate the beads from the solution for 20 s and remove the supernatant.

1.3 Remove the tube from the magnet and resuspend the Protein A/G Magnteic Beads in 150 μl binding buffer. Wash by gentle pipetting, then put the tube back to the magnetic rack for 20 s, and discard the supernatant. Repeat this step for 2 times.

2 Binding of Antibody

2.1 Add your antibody (Ab) (typically 5-10 μg) to 50 μl binding buffer, in order to dilute Ab to the appropriate concentration. The optimal amount of Ab needed depends on the individual Ab used.

2.2 Add Ab mixture to the Protein A/G Magnteic Beads from above. Rotate tube for 1 h at room temperature or 4h at 4°C, then resuspend thoroughly by pipetting up and down.

2.3 Place the tube on the magnet for 20 s and remove the supernatant.

2.4 Remove the tube from the magnet and resuspend the beads-Ab complex in 150 μl binding buffer. Wash by gentle pipetting.

3 Immunoprecipitation of Target Antigen

3.1 Place the tube (from step 2.4 in "Binding of Antibody") on the magnet and remove the supernatant.

3.2 Add your sample containing the antigen (Ag) (typically 100-150μl) and gently pipette to resuspend the Protein A/G Magnteic Beads-Ab complex.

3.3 Incubate with rotation for 10 min at room temperature to allow Ag to bind to the Protein A/G Magnteic Beads-Ab complex.
Note:Depending on the affinity of antibody, it may be necessary to increase the incubation time for optimal binding.

3.4 Place the tube on the magnet. Transfer the supernatant to a clean tube for further analysis, if desired. The supernatant is the non-binding fraction.

3.5 Wash the Magbeads-Ab-Ag complex 3 times using 300 μl binding buffer for each wash. Separate on the magnet between each wash, remove supernatant and resuspend by gentle pipetting .

3.6 Resuspend the Protein A/G Magnteic Beads-Ab-Ag complex in 150 μl binding buffer and transfer the bead suspension to a clean tube. This is recommended to avoid co-elution of the proteins bound to the tube wall.

4 Elution

Add 300 μl wash buffer into the tube vortex or tilt and rotate for 5 min, then put the tube back onto the magnetic rack for 1 min and collect the supernatant into a new tube then add 10-20 μl elution buffer into the collected solution.
The final solution can be used as samples for denaturing SDS-PAGE.

Recommended buffer examples

Important Notes before Beginning

1 Before immunoprecipitation, please be sure to carefully read the operating instructions.
2 This product requires use of a magnetic separator.
3 Protein A/G Magnteic Beads should be suspended uniformly before use.
4 Protein A/G Magnteic Beads should be kept in storage solution and prevent dry.
5 Do not freeze or centrifuge MagBeads protein A/G.
6 In order to ensure the best results, please select an antibody with strong specificity.
7 For the IP experiments, different antibodies and antigens will display different binding affinities. Antibody and antigen binding may be altered based on use of binding buffer and washing buffer. Some operator optimization may be necessary.
8 This product is only intended to be used as directed. All other uses are prohibited.

Figure 1 General Protocol for Immunoprecipitation